The name bikunin was recently proposed to describe a double-headed proteinase inhibitor of the bovine pancreatic trypsin inhibitor (Kunitz) family found in human plasma. Bikunin is associated with three distinct but related heavy chains called HC1, HC2 , HC3 and, possibly, at least one other uncharacterized chain. We have previously determined that bikunin can be linked to HC1 and HC2 to constitute inter-alpha-inhibitor (I-alpha-I) or to HC3 to form pre-alpha-inhibitor (P-alpha-I). Bikunin is also bound to HC2 alone but no specific name has been proposed for this protein. Despite the appreciable concentrations of these proteinase inhibitors in blood, almost nothing is known of their function. We have recently studied the structure of human bikunin proteins, on the basis that this will guide us to a better understanding of their function. I-alpha-I and P-alpha-I resist dissociation in SDS even in the presence of reagents that cleave disulfide bonds, and we have shown that this unusual behavior is due to the presence of a novel type of protein cross-link. The molecular structure of one of these covalent interchain cross-links has recently been determined in our laboratories. We found that the link between HC3 and bikunin is composed of a chondroitin-4-sulfate carbohydrate chain that originates from a typical O-glycosidic link to Ser-10 of bikunin. The heavy chain (HC3) is esterified via the alpha-carbon of its C-terminal Asp to the C-6 of an internal N-acetylgalactosamine of the glycosaminoglycan chain. This is the first description of a covalent glycosaminoglycan-mediated protein cross-link. Both bikunin and the heavy chains are synthesized as larger precursors that undergo proteolytic processing during biosynthesis and chain assembly. The processes by which glycosaminoglycan is able to covalently link proteinase inhibitor bikunin chains to larger polypeptides are unknown. We now have a unique opportunity to study the integrated processing and assembly events involved in the generation of human plasma bikunin proteins and the biogenesis of the novel cross-link that assembles them. This proposal plans to investigate the relationship between structure of these proteins, their assembly, and their activity.